SUMMARY Eukaryotic cells contain within them a myriad of spatially distinct sites that serve a variety of functions. To facilitate this organization, eukaryotic gene expression is routinely spatially regulated through the trafficking and sequestration of thousands of different RNA molecules to distinct cellular locations. Misregulation of this process leads to detrimental phenotypes in a wide range of systems, from developmental defects in Drosophila to neurological disease in humans. Despite this importance, our knowledge of the regulation of RNA localization is quite limited. For other modes of post-transcriptional regulation like splicing, our understanding of how the interactions of RNA binding proteins (RBPs) and RNA motifs lead to specific outcomes is much more mature. This relies on many years of work by many groups that have defined the regulatory language of splicing and allows us to make predictive and combinatorial models about how splicing is regulated across conditions and cellular environments. We lack such an ability with regards to RNA localization, in large part because we lack the analogous ?parts list? that defines the language of localization regulation. Generally, the effect of RBP/RNA binding on post-transcriptional regulatory processes like splicing or RNA decay is consistent across cell types. For example, if an RBP promotes the splicing of an exon in one cell type, it often exerts a similar effect on that exon in another cell type. However, because RNA localization is inherently tied to cell morphology, the generality of localization regulation across cell types is unknown. Combinations of RNA motifs and RBPs that result in RNA localization to projections in neurons are also broadly present in non-neuronal cell types. Are these RNAs trafficked in non-neuronal cells? If so, to where? The answers to these questions first require a better knowledge of the underlying regulatory language of localization. The experiments proposed here are the beginnings of our efforts to define this language and test its generality. We have developed methods to isolate and profile subcellular transcriptomes from the projections of neurons and the apical and basal regions of epithelial cells. We will use these techniques to take a biochemical and transcriptome-wide approach to defining RBP/RNA interactions that regulate localization in two mammalian cell types: neurons and intestinal epithelial cells. By identifying transcripts that are mislocalized in RBP-null cells, we will identify functional RBP/RNA interactions. Using a massively parallel reporter assay, we will take an unbiased approach to finding RNA sequences that regulate localization. By comparing the activities of identified functional RBP/RNA interactions across cell types, we will for the first time be able to directly assess the generality of RNA localization. This methodical and innovative approach is the first step in our efforts to shed light on this fundamental but poorly understood cellular process.